Voltage-gated K+ (Kv) channels are key regulators of neuronal excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, and in modulating the responses to synaptic inputs and synaptic plasticity. Molecular cloning has provided insights into the basis of neuronal Kv channel diversity with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. Accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling neuronal Kv channel expression, distribution and functioning are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of the voltage- gated K+ (Kv) channels underlying the rapidly activating and inactivating, Kv4.2--encoded "A" currents (IA) in cortical pyramidal neurons rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co-immunoprecipitate with Kv4.2 from brain in Kv4.2--encoded IA channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued simultaneously. Specifically, the studies here will test the hypothesis that Nav21 functions to regulate Kv4.2-encoded IA channels (aim #1), rather than, or in addition to, Nav channels (aim #2) in cortical pyramidal neurons and determine directly the role of Nav21.in regulating the firing properties of these cells (aim #2). To achieve these aims, the expression of endogenous Nav21 will be manipulated in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of IA (and Nav) channels in (mouse) cortical pyramidal neurons will be determined. Parallel experiments will be complete on cortical pyramidal neurons from mice (SCN1b-/- ) harboring a targeted disruption of the SCN1b (Nav21) locus. It is anticipated that these studies will provide fundamentally important new insights into the mechanisms that regulate the expression and the functioning of macromolecular neuronal Kv channel complexes. In addition, the results of the studies here will guide future investigates in this and other laboratories focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of neuronal membrane excitability and on defining the functional consequences of mutations in SCNxb subunits linked to derangements in neuronal excitability. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels are key determinants of neuronal membrane excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, the responses to synaptic inputs and synaptic plasticity. Although accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising pore- forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of neuronal Kv channels. Exploiting molecular genetic strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cortical neurons and on testing the novel hypothesis that the Nav2 accessory subunits actually function to regulate Kv channels rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the regulation of neuronal Kv channels and into the molecular mechanisms controlling neuronal functioning and plasticity.